Intestinal mechanosensory neurons ensure relatively constant movement of content from one end of the gut to the other. There are at least three populations of mechanosensory neurons: mechanosensory intrinsic primary afferent neurons that form the afferent limb of intrinsic reflexes; mechanosensory intestinofugal neurons (IFNs) that project their axons outside the intestinal wall to prevertebral ganglia where they make synaptic contact with sympathetic neurons; and mechanosensory colon spinal afferent neurons (SANs) with axon collaterals to prevertebral ganglion neurons. The work proposed in this grant focuses on the latter two populations. The basis for mechanosensitivity in IFNs and colon SANs is not understood. We focus the first part of our work on determining whether the degenerin/epithelial Na+-channel (DEG/ENaC) is a component part of the mechanotransducer in these nerves. We will determine by RT-PCR, immunohistochemistry and confocal 3D imaging if DEG/ENaC subunits are expressed in these nerves and by intracellular and patch clamp recording the role of DEG/ENaC in IFN function. In the second part of our work, we will test our hypothesis that mechanosensory colon SANs with axon collaterals are acid sensitive. DRASIC and the vanilloid receptor (VR1) can be directly activated by protons. We will determine by RT-PCR if DRASIC is present in colon SANs with axon collaterals and by immunohistochemistry whether DRASIC and VR1 are colocalized in these nerves. Next, we will examine the effect of acidic solutions placed in the colon on synaptic input to the release of substance P in prevertebral ganglia, and determine the role DRASIC and VR1 play in acid-evoked responses. The significance of our work is two fold. Firstly, it wil provide data to support the notion that DEG/ENaC cation ion channel bridges the gap between colonic motility and mechanosensitivity in intestinal afferent nerves. Secondly, it will provide data to support the hypothesis that DRASIC and VR1 mediate acid sensitivity in mechanosensitive colon SANs with axon collaterals and that these mechanosensory nerves may be sensitized during inflammation. The basic knowledge derived from our studies is expected to improve the understanding of the functional significance of the molecular components of mechanosensitivity and chemosensitivity of intestinal afferent nerves.